LOCUS       sequence-163057-        6489 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  cLac143; pALM143; IPTG inducible system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6489)
  AUTHORS   Markley AL, Begemann MB, Clarke RE, Gordon GC, Pfleger BF
  TITLE     Synthetic biology toolbox for controlling gene expression in the 
            cyanobacterium Synechococcus sp. strain PCC 7002.
  JOURNAL   ACS Synth Biol. 2015 May 15;4(5):595-603. doi: 10.1021/sb500260k. 
            Epub 2014 Sep 25.
  PUBMED    25216157
REFERENCE   2  (bases 1 to 6489)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1021/sb500260k";
            journalName: "ACS Synth Biol"; date: "2015-05-15- 15"; volume: "4"; 
            issue: "5"; pages: "595-603"
COMMENT     Sequence Label: pACSA_PcptOO_C38T,A39T_YFP_PMB2_LacIWF
FEATURES             Location/Qualifiers
     source          1..6489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(64..83)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     274..295
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        510..614
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(615..683,684..1475)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(833..852)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1646..2234
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2135..2154
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2388..2405
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2522..2543
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2558..2563,2564..2581,2582..2588)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2596..2612
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2620..2636
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2620..2636
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2654..2672
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2654..2671
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    3231..3250
                     /label=lac operator (symmetric)
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     protein_bind    3335..3351
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             3400..3429
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             3442..3453
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             join(3475..3477,3478..3480,3481..4194)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3520..3541)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3781..3800)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4128..4149
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4264..4282)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4278..4325
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        4423..4457
                     /label=BBa_J23110
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.33"
     CDS             4487..5569
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDFSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4506..4525)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     primer_bind     complement(6283..6302)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6284..6302)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6309..6326)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6309..6325)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6318..6340)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      join(6466..6489,1..432)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
       61 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
      121 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
      181 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
      241 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
      301 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
      361 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
      421 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      481 catcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
      541 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
      601 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
      661 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
      721 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
      781 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
      841 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
      901 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
      961 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1021 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1081 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1141 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     1201 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1261 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     1321 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     1381 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     1441 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     1501 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     1561 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     1621 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     1681 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     1741 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     1801 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     1861 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     1921 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     1981 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2041 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2101 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2161 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2221 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     2281 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     2341 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     2401 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     2461 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     2521 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     2581 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     2641 attacgccaa gctatttagg tgacactata gaatactcaa gctatgaggt gactgccgca
     2701 ctcaatgttc aaccagatct agtgattatt tcggggttag cagccgatgc gggcaacctg
     2761 gtgaagcaac tgcgagaatt aggttacaac ggcattattg ttggggggaa cggcttaaat
     2821 acttctaata ttttccccgt ctgccaagca aaatgtgatg gggtgttggt ggcccaagcc
     2881 tacagtgccg agttagataa tgagattaac cgcgcgtttc gggacgccta ttttcaacaa
     2941 aaccaaaaag agccgcccca atttagtgcc caggctttta cggcgatcca agtttttgtt
     3001 gaagccctca gcagcctcga tgaaaaaacg cccttagaaa ctcttgctct accggacttg
     3061 cgacgacaac tgcgggacga aatttttgca ggtacctacg tcacgccttt gggtgaaatt
     3121 tccttcacag aggaagggga aattgtccag aaggaatttt ttgtggccca aattgaaatg
     3181 gatgaatcgg gtcaacaggg gcgtttcgcc ttcattgaaa cgaacaaacg aattgtgagc
     3241 gctcacaatt cggaattctt aacaaaaaag caggaataaa attaacaaga tgtaattgac
     3301 ataagtccca tcaccgttgt ataaatgtgt ggaattgtga gcggataaca atttcacaca
     3361 ccaactcata aagtcaagta ggagattaat tccatgggcc atcatcatca tcatcatcat
     3421 catcatcaca gcagcggcca tatcgaaggt cgtcatatgc aggcagcagc ctcgatggtg
     3481 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     3541 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     3601 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     3661 accaccttcg gctacggcct gcagtgcttc gcccgctacc ccgaccacat gaagcagcac
     3721 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     3781 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     3841 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     3901 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     3961 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     4021 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     4081 agctaccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     4141 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaacgaggg
     4201 cggtgctttg gcaggatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     4261 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgacgggtt
     4321 ttttgtctag atcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca
     4381 taagggagag cgtcgagatc ccggacacca tcgaatggcg catttacagc tagctcagtc
     4441 ctaggtacaa tgctagccgg aggagggtca attcatggtg gtgaatgtga aaccagtaac
     4501 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     4561 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     4621 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     4681 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     4741 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     4801 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     4861 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     4921 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga
     4981 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     5041 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     5101 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactttagtg ccatgtccgg
     5161 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     5221 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     5281 tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc
     5341 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     5401 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
     5461 aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     5521 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     5581 ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct
     5641 tcaacccagt cagctccttc cggttcgcgc tcaagttgat cgaatgtatg ccccggatat
     5701 gacctaggga gattcttcta aagcgagcag ggtttgccaa gtggcgatcg cctcagggaa
     5761 ccataaccaa ctttgttcga ggcggcgggg gttgagctgg gaacgatcga gatctagggc
     5821 gatcgcccgg agtttttcgg ctttccctac aagaatttcc tggtcaacgt cggtgggttg
     5881 cctttgggcc aaatcgtaca tactaatcgc caggcccgca taaatctccg cctggagggc
     5941 tggggggaaa tcctgggtcg ttgccgccgc taaggcttca taccaacgat cattggcggc
     6001 ttcgtaatct gcattgaggt aatggacaaa acccagggcc atcaaaattt cggggtcgtt
     6061 gggctgttgg gctagggcca tgtcccaatt gtcttgcacc gtggcgatcg ccgttgcttg
     6121 ggttggcgat ggtgggacgg cagccaaagt tgtttgccag cgatggtggg acggcagcca
     6181 aagttgtttg ccagcgatgg tgggacggca gccaaagttg tttgccaggt gactgccgca
     6241 ctcaatggtg ggacggcagc caaagttgtt tgccagaatt cgccctatag tgagtcgtat
     6301 tacaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     6361 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     6421 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg
     6481 gcgcattaa
//
